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AUGUST: WEEK 4
August, 22nd
Streak of E17-2, E18-2, J101-31, J101-E5 and ENTERO-4C5 on LB agar + Cm12.5 plate to test Plux promoter with different RBSs.
August, 23rd
T9002-ENTERO and ENTERO-RBS were diluted 1:500 in a final volume of 13 ml and 1 ml respectively of M9 + Amp + Cm12.5.
August, 24th
E43 ligation was transformed in 100 μl of MGZ1 competent cells.
August, 25th
Three colonies were picked from E43 plate.
Every plasmid was digested with EcoRI and PstI resctriction endonucleases. A medium size and a small size agarose-gel were prepared and, after three hours digestion, gel-electrophoresis was carried out: As showed in images E36 did not show the correct insert band while J101-31, J101-E5 and J101-E7 showed light intensity bands as they were in a low copy number plasmid. The correct parts were gel-extracted and DNA was quantified:
BBa_R0040 in J61002 was inoculated in 5 ml LB + Amp while J101-31, J101-E5 and J101-E7 were again inoculated in 8 ml of LB + Cm12.5 for plasmid purification.
August, 26th
Glycerol stock for E43-3 was prepared according to protocols.
At this point, probably E43-2 and E43-3 tubes were confused! Anyway these plasmids were digested:
A medium size agarose-gel was prepared. BBa_R0040 in J61002 resulted incorrect, while other bands had a light intensity but we decided however to gel-extract and ligate them:
E24-2, E25-1, E26-2, E27-2 and RBS32 were inoculated in 1 ml of M9 + Amp to test AiiA efficiency in high copy number plasmids, taking cultures supernatants with a protocol similar to the one used for low copy number vectors. August, 27th
E24-2, E25-1, E26-2, E27-2 and RBS32 were diluted 1:500 in a final volume of 6 ml of M9 + Amp; a tube with 6 ml of M9 + Amp medium was also prepared. These six tubes were grown for 2 hours; then 3OC6-HSL was added at a final concentration of 100 nM. Supernatants were taken at the moment of supplementation (t = 0 h), after 1 hour, after 2 hours and after 4 hours; then they were stored at -20°C.
August, 28thInoculum of T9002-ENTERO and ENTERO-RBS in 1 ml of M9 + Amp + Cm12.5 to test AiiA efficiency in high copy number plasmids with the suprnatants collected on August, 27th.
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Team:UNIPV-Pavia/Calendar/August/week4
From 2011.igem.org
(Difference between revisions)
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<tr> | <tr> | ||
<td class="row"><b>E2 in pSB1C3</b></td> | <td class="row"><b>E2 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1</td> | <td class="row">1</td> | ||
<td class="row">E2-2 (E-P)</td> | <td class="row">E2-2 (E-P)</td> | ||
Line 453: | Line 453: | ||
<tr> | <tr> | ||
<td class="row"><b>E3 in pSB1C3</b></td> | <td class="row"><b>E3 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1.5</td> | <td class="row">1.5</td> | ||
<td class="row">E3-1 (E-P)</td> | <td class="row">E3-1 (E-P)</td> | ||
Line 463: | Line 463: | ||
<tr> | <tr> | ||
<td class="row"><b>E4 in pSB1C3</b></td> | <td class="row"><b>E4 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1.5</td> | <td class="row">1.5</td> | ||
<td class="row">E4-2 (E-P)</td> | <td class="row">E4-2 (E-P)</td> | ||
Line 473: | Line 473: | ||
<tr> | <tr> | ||
<td class="row"><b>E5 in pSB1C3</b></td> | <td class="row"><b>E5 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1.5</td> | <td class="row">1.5</td> | ||
<td class="row">E5-2 (E-P)</td> | <td class="row">E5-2 (E-P)</td> | ||
Line 483: | Line 483: | ||
<tr> | <tr> | ||
<td class="row"><b>E6 in pSB1C3</b></td> | <td class="row"><b>E6 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1</td> | <td class="row">1</td> | ||
<td class="row">E6-1 (E-P)</td> | <td class="row">E6-1 (E-P)</td> | ||
Line 493: | Line 493: | ||
<tr> | <tr> | ||
<td class="row"><b>E7 in pSB1C3</b></td> | <td class="row"><b>E7 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">2</td> | <td class="row">2</td> | ||
<td class="row">E7-2 (E-P)</td> | <td class="row">E7-2 (E-P)</td> | ||
Line 503: | Line 503: | ||
<tr> | <tr> | ||
<td class="row"><b>E9 in pSB1C3</b></td> | <td class="row"><b>E9 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1.5</td> | <td class="row">1.5</td> | ||
<td class="row">E9-2 (E-P)</td> | <td class="row">E9-2 (E-P)</td> | ||
Line 513: | Line 513: | ||
<tr> | <tr> | ||
<td class="row"><b>E10 in pSB1C3</b></td> | <td class="row"><b>E10 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1</td> | <td class="row">1</td> | ||
<td class="row">E10-1 (E-P)</td> | <td class="row">E10-1 (E-P)</td> | ||
Line 523: | Line 523: | ||
<tr> | <tr> | ||
<td class="row"><b>E11 in pSB1C3</b></td> | <td class="row"><b>E11 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1.5</td> | <td class="row">1.5</td> | ||
<td class="row">E11-1 (E-P)</td> | <td class="row">E11-1 (E-P)</td> | ||
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<tr> | <tr> | ||
<td class="row"><b>J101-31 in pSB1C3</b></td> | <td class="row"><b>J101-31 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1</td> | <td class="row">1</td> | ||
<td class="row">J101-31 (E-P)</td> | <td class="row">J101-31 (E-P)</td> | ||
Line 543: | Line 543: | ||
<tr> | <tr> | ||
<td class="row"><b>J101-E5 in pSB1C3</b></td> | <td class="row"><b>J101-E5 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1</td> | <td class="row">1</td> | ||
<td class="row">J101-E5 (E-P)</td> | <td class="row">J101-E5 (E-P)</td> | ||
Line 553: | Line 553: | ||
<tr> | <tr> | ||
<td class="row"><b>J101-E7 in pSB1C3</b></td> | <td class="row"><b>J101-E7 in pSB1C3</b></td> | ||
- | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part: | + | <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB1C3">pSB1C3</a> (E-P)</td> |
<td class="row">1</td> | <td class="row">1</td> | ||
<td class="row">J101-E7 (E-P)</td> | <td class="row">J101-E7 (E-P)</td> |
Revision as of 08:16, 16 September 2011